1. Technical Field
The invention is in the technical field of the purification of rebaudioside A (also known herein as “Reb A” and “RA”) from crude extracts from the plant Stevia rebaudiana, L. Bertoni (“Stevia”). Reb A is a diterpene glycoside found, along with many other glycosides, sterebins, and other compounds, in Stevia extracts. Reb A is the sweetest tasting (roughly 250 to 450 times the sweetness of sucrose at sweetener concentrations used in comestibles) glycoside in Stevia and has enormous commercial potential as a non-caloric sweetener. Generally speaking, the Stevia glycosides, except rebaudioside A, have an undesirable aftertaste (some have a bitter aftertaste), which generally limits the use of such non-Reb A glycosides as sweeteners. Moreover, sterebins (a group of diterpene compounds commonly known as “yellow oil”) in Stevia extracts have an extremely bitter taste, even in minute concentrations. In short, the technical field, and the technical problem, is to isolate, especially at industrial scale, 99+% purity Reb A from crude Stevia extract; such purity eliminates the bitterness or off-taste that characterizes all currently available Stevia sweeteners.
2. Related Art
The six rebaudiosides (A to F), stevioside (the predominant glycoside in extracts from wild type Stevia), dulcosides, and sterebins are difficult to separate in solution. In particular, the six rebaudiosides share the same diterpene skeleton and differ only in glycoside moieties; all but Reb A has some degree of bitter aftertaste. Scores of patents and journal articles describe efforts to produce pure (i.e., 99+% purity) rebaudioside A, but to date no method of producing pure Reb A in the lab, much less in commercial quantities, has been reported. Many of the reported methods of production require the use of ion exchange columns or gases, the economics of which undermine scale-up to the commercial quantities of sweetener required for the beverage, packaged food, baking, candy, personal care product, pharmaceutical, and other industries.
Most methods for extraction and purification of diterpene glycosides from Stevia use complicated processing of crude extracts and require high concentrations of rebaudioside A in starting materials, yet have low yields and low purifies (<95%) of rebaudioside A. Some methods use alkanols and/or hydrous alkanols as solvents. For instance, in paragraphs 49 to 53 of U.S. Published Patent Application No. 2003/0138538 A1 of Kitazume, et al., a method is described whereby plants or dried leaves from Stevia rebaudiana are processed using methanol, hydrous methanol, or hydrous ethanol (but not using a reflux process) and column purification to produce an extract containing a minimum of 40% rebaudioside A by weight and with a rebaudioside A to stevioside ratio is 1.5:1. In Kitazume's method, if the starting plant material is not 40% rebaudioside A by weight and with a rebaudioside A to stevioside ratio of 1.5:1 (“Kitazume Threshold Purity”), column purification or recrystallization must be used to produce rebaudioside A of sufficient purity needed to support the object of the Kitazume patent application (the substitution on rebaudioside A of β-1,4-galactosyl using a β-1,4-galactosyl transferase enzyme). The preferred starting material in Kitazume is a Stevia extract with at least 70% Reb A content. In the Kitazume method, when recrystallization is used to produce rebaudioside A of Kitazume Threshold Purity for the transferase reaction, the Stevia extract is dissolved to saturation in a hydrophilic organic solvent, such as methanol or ethanol, with no water in the solvent, and the solution concentrated or cooled, then filtered to collect the precipitate, a higher purity rebaudioside A; process parameters, e.g., heating and cooling rates, filtration specifications, etc., of the purification of Reb A are not disclosed. Kitazume also does not disclose the purity of the Reb A produced by recrystallization, only that recrystallization can be used when the starting material is below Kitazume Threshold Purity. Importantly, in Kitazume, all Reb A material is produced using cation-exchange resin and anion-exchange resin steps (paragraph 50) following any crystallization or recrystallization steps. A Reb A purity higher than 91.3% (Example 3) is not disclosed, nor is the solubility of Kitazume's Reb A disclosed. In fact, Kitazume does not disclose whether the 91.3% purity Reb A used in the Examples was produced using recrystallization or only by column purification. Reb A with 91.3% purity may be acceptable for the transferase reaction, but it is not acceptable for commercial uses in foods and beverages.
U.S. Pat. No. 5,962,678 to Payzant et al., describes a seven-step extraction and purification of Reb A from Stevia plant material, including processing through two ion exchange columns and precipitation out of a methanol solution. For purification of Reb A, after removing mixed sweet glycosides from the second ion exchange column with methanol, the collected eluent is dried. Upon refluxing the dried solids in a methanol solution and then cooling the solution, stevioside precipitates and is captured by filtration. The filtrate is then concentrated (presumably by evaporating the solvent) and cooled and rebaudioside A precipitates. The method in Example 5 of Payzant is described as yielding rebaudioside A with purity up to 98.6%. However, using 80% purity Reb A Stevia extract as starting material, repeated efforts by the inventors of the present invention to replicate Payzant's purification of Reb A failed; typical results were a Reb A yield of less than 5% by weight and amorphous crystals with a noticeable, bitter aftertaste. Attempts by a third party to replicate Payzant also failed (unpublished data). In attempts to replicate Example 5, precipitation of Reb A did not begin until more than 40 hours after heat was removed from the methanol solution. The Payzant purification method in Example 5 apparently requires Reb A starting material obtained by following the steps in Payzant claim 1 (the seven-step process using methanol) rather than by procuring commercially available Stevia extracts. The Payzant method, therefore, is neither generally applicable to Stevia extracts nor scaleable to commercial production of Reb A.
In addition to not being replicable, Payzant teaches away from using hydrous solvents. Payzant uses anhydrous methanol as a reflux solvent to purify stevioside. In Example 2, Payzant states, “Since the sweet glycosides were absorbed on to the XAD-7 resin from water, there is water contained within or on the surface of this resin and this water is removed with the methanol. The only reason for going to dryness is to remove this water. If the minor amount of water wasn't there or if it were removed by the some other means, then the methanol could be removed by evaporation to some level and then the sweet glycosides would crystallize from the solution.” In Example 3, Payzant states, “By being anhydrous or as free from water as possible the crystalline yield is maximized and the crystallization time is minimized. For example, 0.5% water in the methanol in this initial crystallization will significantly reduce the yield. The Stevioside can be further purified, if desired, by using the purification technique set forth in Example 5 below.” In Example 5, Payzant discloses a purification in which a Stevia extract containing 90.2% Reb A (in Example 3, however, the final product had 7.9% Reb A content) is refluxed in a solvent of 86.67% methanol and 13.33% water by mass; this procedure is said to yield 98.6% Reb A; heating rate, cooling rate, stirring, and temperatures are not disclosed. Payzant notes that the extract used in Example 5 is “not from Example 4”, but elsewhere discloses achieving only 7.9% purity Reb A. Payzant states in Example 5 that including water in the solvent is “a convenience in the laboratory”, but “on a commercial scale anhydrous solvent might be more convenient”. In Examples 3 and 5, Payzant expressly teaches away from using hydrous alkanol solvents for purification of Stevia glycosides. Payzant does not disclose how the 90.2% purity Reb A used in Example 5 was obtained, i.e., the method of improving Reb A purity from 7.9% to 90.2% is not enabled, and therefore Example 5 is not enabled. Payzant also does not disclose the method used to determine the Reb A purities claimed. Under the Reb A reference standards currently available from Chromadex (www.chromadex.com) or Wako (www.wako-chem.co.jp), Payzant's claimed purities are thought to be overstated. As noted above, Payzant's Reb A purification method failed when existing commercial quality Stevia extracts were used as starting material. The Payzant method of purifying Reb A requires first precipitating out stevioside in a methanol reflux, and Payzant should be so limited as a reference. Putting aside the omission of how 90.2% Reb A purity was obtained, the lack of a reference standard, and the instructions to use anhydrous solvents, the process complexity, low throughput, and low yield in the Payzant method mean that it cannot be used as an industrial process. Moreover, the use of a methanol reflux in isolating Reb A, with concomitant risk of methylated contaminants in the final product, may present a barrier to obtaining “generally recognized as safe” status with government regulators of food products.
The existing art of producing higher purity rebaudioside A uses elaborate sequences of ion exchange columns and other low-throughput procedures. The existing methods used by Kitazume, Payzant, and others do not produce 99+% purity Reb A, whether by crystallization, recrystallization, column purification, or combinations thereof. There are heretofore unsolved problems that have caused unacceptably high levels of contamination in Reb A final products. The existing art in the industrial production of Stevia glycosides, including rebaudioside A, typically starts with Stevia plant material and produces a Stevia extract that has 40% to 90% purity Reb A mixed with various Stevia glycosides and “yellow oil”; such extracts are commonly called “Stevia extract powder” or “Stevia extract”, and are herein called “Stevia starting material”. Stevia starting material is abbreviated “SSM” in the Drawings. The lack of an industrial method of producing 99+% purity rebaudioside A has meant that Stevia sweeteners are today typically used (e.g., in pickling) where other flavourants mask the bitter or astringent aftertastes arising from residual contaminants in the Stevia sweeteners. Even 1% contamination by other Stevia compounds produces a noticeable, bitter aftertaste in Reb A sweeteners, which led Dobberstein, in U.S. Pat. No. 4,612,942, to limit use of Stevia glycosides in orally consumable compositions to a level (the “sweetness level threshold”) that modified the taste of the compositions, but could not be perceived as sweet. The term “orally consumable composition”, as used herein, means substances that are contacted with the mouth of a human or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested. There is a need for non-caloric materials that can sweeten, modify, or enhance the flavor of orally consumable compositions such as foodstuffs, smoking compositions, chewing compositions, oral hygiene compositions, and medicinal compositions to improve or vary the sensory perceptions thereof.
In view of the enormous market for a non-caloric, natural sweetener for use in foodstuffs, beverages, medicines, tobacco products, candies, etc., a method of obtaining 99+% purity rebaudioside A has long been sought. 99+% purity Reb A is useful as a reagent, as an ingredient in foods and beverages, as a standalone sweetener, and as a co-sweetener. There is an unmet demand for (1) a method of producing 100% pure, water soluble, rebaudioside A in the lab and (2) a method of producing industrial quantities of 99+% purity, water soluble, rebaudioside A suitable for use as a reagent, as an ingredient in foods and beverages, as a standalone sweetener, and as a co-sweetener. It will be shown that, counter to Payzant's teaching, having a significant water content in the reflux solvent is essential to producing 99+% purity Reb A and to avoiding the contamination reported by all other researchers.